Isolation of a complementary bacteriophage from a phage-resistant mutant expands control strategies against Xanthomonas campestris.

Authors

Qing Yu, Qingshan Wu, Tao Lu, Ruina Yang, Zheng Fang, Lan Xiang, Qiuping Liu, Leitao Tan, Xiaosheng Zhao, Chuangen Lin, Qingbei Weng

Published in

Frontiers in microbiology. Volume 17. Pages 1853113. Epub Aug 03, 2026.

Abstract

Bacterial resistance to bacteriophages represents a major limitation for the durable use of phage-based biocontrol strategies in agriculture. Here, we used a phage-resistance derivative of Xanthomonas campestris pv. campestris (Xcc) as a selective host to isolate an additional phage with activity against resistant bacteria. A spontaneous mutant, designated Xcc 8004R, was isolated following exposure of Xcc 8004 to phage X1. Xcc 8004R exhibited a growth profile comparable to that of the wild-type strain, but differed in colony appearance, X1 adsorption efficiency, and motility phenotypes. Whole-genome resequencing identified mutations in genes annotated as encoding a lipopolysaccharide (LPS) core biosynthesis protein, PilY1, N-acetylmuramoyl-L-alanine amidase, and IS1404 transposase, and a conserved hypothetical protein. Using Xcc 8004R as the isolation host, we recovered a bacteriophage, vB_Xcc_GYRb1 (GYRb1), from agricultural soil. GYRb1 infected both Xcc 8004 and Xcc 8004R and exhibited different adsorption kinetics on the two strains. Transmission electron microscopy showed an icosahedral head and a long, non-contractile tail. Genome sequencing revealed a circularly assembled 91,478-bp double-stranded DNA genome encoding 128 predicted open reading frames and two tRNAs. No recognizable antibiotic resistance or bacterial virulence genes were detected. Phylogenomic, ANI, and gene-sharing network analyses suggest that GYRb1 is highly divergent from currently available related phages and may represent a candidate genus-level lineage within the class Caudoviricetes. GYRb1 suppressed the growth of Xcc 8004 and Xcc 8004R in vitro and reduced disease severity in cabbage leaf. A cocktail containing GYRb1 and X1 reduced OD₆₀₀-based bacterial regrowth compared with single-phage treatments in vitro. Together, these findings support the feasibility of using phage-resistant bacterial mutants as selective hosts to obtain complementary candidate phages, while highlighting the need for receptor identification, resistance-frequency analysis, lysogeny assessment, and broader host-range testing before practical application.

PMID:
42609314
Bibliographic data and abstract were imported from PubMed on 18 Aug 2026.

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